The cellular responses to DNA damage are controlled by checkpoint pathways, which are highly conserved from yeast to human. The long-term goal of this project is to define the regulatory mechanism of the ATR family protein that controls the DNA damage checkpoint. The ATR protein kinase interacts with a partner protein, ATRIP, and acts in the form of the ATR-ATRIP complex. In budding yeast, Mec1 and Ddc2 correspond to ATR and ATRIP, respectively. Damaged DNAs have to be processed by DNA modification enzymes for proper DNA repair. Generation of single-strand DNA (ssDNA) is one of key steps at the early damage processing. Generated ssDNA at DNA lesions are covered with replication protein A (RPA), which mediates various DNA repair pathways. The ATR-ATRIP/Mec1-Ddc2 complex interacts with RPA-covered ssDNA and accumulates at sites of DNA damage. In budding yeast, Mec1 phosphorylates the Rad9 checkpoint mediator at sites of DNA damage. Phosphorylated Rad9 interacts with the Rad53 kinase, and the Rad9-Rad53 interaction increases Rad53 kinase activity. Activated Rad53 further phosphorylates the target proteins and relays checkpoint signals to the downstream. Current studies thus have provided a clear outline of how Mec1 initiates the phosphorylation cascade. However, the regulatory mechanism has not been fully understood yet. The experiments in this proposal will aim to uncover how Mec1 is activated at sites of DNA damage (Aim 1) and how phosphatases counteract the Mec1- Rad53 phosphorylation cascade (Aim 2). Telomeres are distinguished from DNA breaks that activate the Mec1 checkpoint pathway. The experiments will also aim to define how telomeres inhibit the Mec1 checkpoint pathway (Aim 3). Failure of proper checkpoint activation causes chromosome instability, which may result in cancer development in human. A better understanding of the checkpoint control should lead to better treatment and prevention of cancer.